The goal of the proposed project is to establish the role of the circadian system in the prenatal effects of cocaine exposure during a critical period of embryonic brain development in a diurnal vertebrate, zebrafish. These studies will be based on the results of the previous 3-year funding period, which established a reciprocal relationship between the cocaine and zebrafish clock system, found that melatonin can counteract the effects of cocaine on brain development, identified two strains with contrasting effects of cocaine on circadian system and behavioral effects of cocaine, and characterized the behavioral and molecular effects of cocaine and cocaine withdrawal in adult and aged zebrafish of both genders. The continuation of this research will specifically focus on characterization of the effects of cocaine and circadian clock system on brain development, and address the hypothesis that reciprocal interaction between the two is due to their contrasting effects on neuronal proliferation during early stages of brain formation. The Specific Aims will address: 1) In-depth characterization of the effects of early cocaine and melatonin exposure on zebrafish brain development and gene expression; 2) An impact of the earlier identified genetic variation in circadian response to cocaine on the effects of prenatal cocaine and melatonin exposure; 3) The role of circadian deficits in the prenatal brain development and effects of cocaine, and whether exogenous melatonin can counteract these effects. Zebrafish will be exposed to cocaine during the major periods of cortical (pallium) and limbic system (dorsomedial and dorsolateral telencephalon) development, synchronized with either the nighttime or daytime circadian phase. Melatonin will be administered prior to cocaine at different circadian phases, in embryos with normal and deficient circadian system. The brain size, cell division rate, dopamine levels, expression of the circadian genes and those encoding for the melatonin receptors, dopamine transporter, cyclin-dependent kinases, cyclin A and growth hormone will be evaluated throughout development and maturation. The time of development and integrity of the neuronal ensembles will be evaluated in transgenic fish with fluorescent markers for alpha-1-tubulin and f-spondin. The cocaine-induced changes in the locomotor activity and sleep will be documented during early post-natal period. Understanding the role of the circadian factors in brain development and mechanisms through which they attenuate prenatal effects of cocaine, should help to design preventive and therapeutic strategies to counteract prenatal cocaine impact on early brain development or subsequent changes in brain functions.